Taste is the essential sensory modality that guides nutrient detection and recognition and toxin rejection. However, a number of health disorders, including obesity and hypertension, can be attributed to taste-guided behaviors. Understanding the neural circuits responsible for taste processing is crucial to developing effective treatment approaches and therapies to combat these disorders. Gustatory information from the oral cavity is transmitted to the rostral nucleus of the solitary tract (rNST) where it is then distributed alog two major projection pathways: the ascending pathway (projecting to the parabrachial nucleus and on to forebrain regions), which regulates stimulus identification and hedonics, and the descending pathway (projecting to the reticular formation and on to oromotor nuclei), which regulates reflexive oromotor behaviors. Despite the integral role of the rNST in taste information distribution, very little is known about intrinsic rNST circuit organization, especially in relatio to these functionally distinct projection pathways. This proposal addresses this knowledge gap using simultaneous multiple in vitro patch clamp recordings and optogenetic, laser- scanning photostimulation to investigate the existence and organization of sub-circuits that are presynaptic to the projection neuron populations. Synchronized synaptic events that originate from shared connections coordinate the activity of neural ensembles, and result in functional sub-circuits. In Aim #1, wild-type C57BL/6 mice will receive injections of different fluorescent retrograde tracers into the parabrachial nucleus and the reticular formation, labeling ascending and descending projection neurons, respectively. After survival for tracer transport, acute rNST slices will be prepared from these animals and 3-4 projection neurons will be simultaneously patch clamped. Spontaneous and evoked synaptic activity will be recorded and analyzed for the presence of correlated synaptic activity indicative of rNST sub-circuits associated with each neuron population. In Aim #2, mice expressing channelrhodopsin under the control of the vesicular GABA transporter will be used to selectively stimulate inhibitory neurons. Both projection neuron populations will be pre-labeled as in Aim #1, and 3-4 projection neurons will be simultaneously patch clamped. A focal 473 nm laser spot will be systematically scanned over the surface of the slice while the resulting inhibitory synaptic responses are recorded. This optogenetic approach will produce spatial distribution maps of the local inhibitory interneurons presynaptic to a given projection neuron, which will then be compared between and within neuron populations. These maps will reveal differences between projection neuron populations in the spatial and inter-subdivision extent of inhibitory presynaptic connectivity, and identify inhibitory sub-circuits within rNST. This project will not only provide valuable information on gustatory sensory processing within the brainstem, but also on the differential role of local circut interactions within functionally distinct taste pathways.